LOCUS       sequence-215805-        8020 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of the CDK4 protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8020)
  TITLE     Salmon Lab plasmids
REFERENCE   2  (bases 1 to 8020)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pCLX-PGK-hCDK4
FEATURES             Location/Qualifiers
     source          1..8020
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     LTR             385..642
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    689..814
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1306..1423
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_feature    1783..2016
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2201..2245
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     protein_bind    2522..2542
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        2547..3057
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2976..2994
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     protein_bind    3058..3082
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4000..4024)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    4042..4630
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4095..4115)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4513..4524)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(4773..4806)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     LTR             4826..5006
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5044..5063)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5109..5164
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5163..5182)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5523..5539)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5523..5539)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5536..5558)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5547..5563
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5571..5577,5578..5595,5596..5601))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5616..5637
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5696..5891
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5742..5877
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5897..6031
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5934..5953)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5988..6007
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6099..6116)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6270..6858)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6350..6369)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7029..7820,7821..7889))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7652..7671
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7890..7994)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggaggcg tggcctgggc gggactgggg agtggcgagc
      421 cctcagatcc tgcatataag cagctgcttt ttgcctgtac tgggtctctc tggttagacc
      481 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
      541 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
      601 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
      661 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
      721 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
      781 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
      841 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
      901 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
      961 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     1021 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     1081 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaaaaa
     1141 agcacagcaa gcagcaggat cttcagacct ggaaattccc tacaatcccc aaagtcaagg
     1201 agtagtagaa tctatgaata aagaattaaa gaaaattata ggacaggtaa gagatcaggc
     1261 tgaacatctt aagacagcag tacaaatggc agtattcatc cacaatttta aaagaaaagg
     1321 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     1381 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     1441 cagcagaaat ccactttgga aaggaccagc aaagctcctc tggaaaggtg aaggggcagt
     1501 agtaatacaa gataatagtg acataaaagt agtgccaaga agaaaagcaa agatcattag
     1561 ggattatgga aaacagatgg caggtgatga ttgtgtggca agtagacagg atgaggatta
     1621 gaacatggaa aagtttagta aaacaccata aggaggagat atgagggaca attggagaag
     1681 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1741 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1801 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1861 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1921 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1981 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     2041 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     2101 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     2161 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     2221 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     2281 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     2341 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     2401 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     2461 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgctagt gattatcgga
     2521 tcaactttgt atagaaaagt tgccacgggg ttggggttgc gccttttcca aggcagccct
     2581 gggtttgcgc agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac
     2641 cctgggtctc gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc
     2701 cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg
     2761 ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac
     2821 ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc
     2881 tgctcagcgg ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt
     2941 ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg
     3001 gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccaggca
     3061 agtttgtaca aaaaagcagg cttcaccatg gctacctctc gatatgagcc agtggctgaa
     3121 attggtgtcg gtgcctatgg gacagtgtac aaggcccgtg atccccacag tggccacttt
     3181 gtggccctca agagtgtgag agtccccaat ggaggaggag gtggaggagg ccttcccatc
     3241 agcacagttc gtgaggtggc tttactgagg cgactggagg cttttgagca tcccaatgtt
     3301 gtccggctga tggacgtctg tgccacatcc cgaactgacc gggagatcaa ggtaaccctg
     3361 gtgtttgagc atgtagacca ggacctaagg acatatctgg acaaggcacc cccaccaggc
     3421 ttgccagccg aaacgatcaa ggatctgatg cgccagtttc taagaggcct agatttcctt
     3481 catgccaatt gcatcgttca ccgagatctg aagccagaga acattctggt gacaagtggt
     3541 ggaacagtca agctggctga ctttggcctg gccagaatct acagctacca gatggcactt
     3601 acacccgtgg ttgttacact ctggtaccga gctcccgaag ttcttctgca gtccacatat
     3661 gcaacacctg tggacatgtg gagtgttggc tgtatctttg cagagatgtt tcgtcgaaag
     3721 cctctcttct gtggaaactc tgaagccgac cagttgggca aaatctttga cctgattggg
     3781 ctgcctccag aggatgactg gcctcgagat gtatccctgc cccgtggagc ctttcccccc
     3841 agagggcccc gcccagtgca gtcggtggta cctgagatgg aggagtcggg agcacagctg
     3901 ctgctggaaa tgctgacttt taacccacac aagcgaatct ctgcctttcg agctctgcag
     3961 cactcttatc tacataagga tgaaggtaat ccggagtgaa cccagctttc ttgtacaaag
     4021 tggtgataat cgaattccga taatcaacct ctggattaca aaatttgtga aagattgact
     4081 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     4141 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     4201 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     4261 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     4321 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     4381 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg
     4441 acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     4501 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     4561 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     4621 gcctccccgc atcgggaatt cccgcggttc gctttaagac caatgactta caaggcagct
     4681 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     4741 cgaagacaag atgggatcaa ttcaccatgg gaataacttc gtatagcata cattatacga
     4801 agttatgctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
     4861 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     4921 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     4981 ttagtcagtg tggaaaatct ctagcagcat ctagttgaat cgccagtgtg tcgagctagc
     5041 agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac
     5101 ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct
     5161 ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt
     5221 ttagagtttg gcaacatatg ccatatgctg gctgccatga acaaaggtgg ctataaagag
     5281 gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga
     5341 cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa
     5401 aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca
     5461 tagctgtccc tcttctctta tgaagatccc tcgacctgca gcccaagctt ggcgtaatca
     5521 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     5581 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     5641 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc
     5701 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     5761 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     5821 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     5881 gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     5941 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     6001 actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg
     6061 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     6121 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     6181 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     6241 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6301 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6361 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6421 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg
     6481 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6541 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6601 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6661 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     6721 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6781 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6841 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6901 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6961 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     7021 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     7081 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     7141 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     7201 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     7261 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     7321 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7381 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7441 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7501 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7561 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7621 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7681 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7741 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7801 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7861 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7921 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7981 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
//
